Photochemical and biological dual-effects enhance the inhibition of photosensitizers for tumour growth

Photosensitizers typically rely on a singular photochemical reaction to generate reactive oxygen species, which can then inhibit or eradicate lesions. However, photosensitizers often exhibit limited therapeutic efficiency due to their reliance on a single photochemical effect. Herein, we propose a new strategy that integrates the photochemical effect (type-I photochemical effect) with a biological effect (proton sponge effect). To test our strategy, we designed a series of photosensitizers (ZZ-sers) based on the naphthalimide molecule. ZZ-sers incorporate both a p-toluenesulfonyl moiety and weakly basic groups to activate the proton sponge effect while simultaneously strengthening the type-I photochemical effect, resulting in enhanced apoptosis and programmed cell death. Experiments confirmed near-complete eradication of the tumour burden after 14 days (Wlight/Wcontrol ≈ 0.18, W represents the tumour weight). These findings support the notion that the coupling of a type-I photochemical effect with a proton sponge effect can enhance the tumour inhibition by ZZ-sers, even if the basic molecular backbones of the photosensitizers exhibit nearly zero or minimal tumour inhibition ability. We anticipate that this strategy can be generalized to develop additional new photosensitizers with improved therapeutic efficacy while overcoming limitations associated with systems relying solely on single photochemical effects.


Experimental procedures 1. Materials and instruments
All reagents used in the synthesis and purification of photosensitive dyes (ZZ-EDA and ZZ-NN) and their intermediates (ZZ-Br, ZZ-NH 2 and ZZ) were of analytical grade.Column chromatography using silica gel (200-300 mesh) was used to purify the compounds.ZZ-EDA (6.0 mM) and ZZ-NN (6.0 mM) were used as stock solutions for all spectroscopic and cell imaging experiments, respectively.The buffer solution used for the optical experiments of the photosensitive dyes in vitro was different pH phosphate buffer saline (PBS) and organic solvent.Commercial probes Lyso-Tracker Red for co-location imaging experiments and MitoProbe DiIC1(5) for mitochondrial membrane potential were purchased from Thermo Fisher Scientific Company (U.S.A.).Calcein-AM/PI Double Stain Kit Calcein-AM/PI for apoptotic experiments was purchased from Yeasen Biotechnology (Shanghai) Co.,Ltd.Photosensitive dyes and its intermediates were characterized using an Avance 600 MHz spectrometer (Bruker Co., LTD Switzerland).High resolution mass spectrometry (Water SYNAPT G2-Si, England) was used to detect the molecular mass.An ultraviolet spectrophotometer (GBC Scientific Equipment Pty LTD, Australia) and fluorescence spectrophotometer FluoroMax-4 (HORIBA, Japan) were used for the spectrometric measurements in vitro.
Edinburgh -Steady State/Transient Fluorescence Spectrometer FLS1000 (Edinburgh, Britain) was used for the fluorescence and phosphorescence lifetime.The cell imaging experiments were completed using a FV1200 spectral confocal multiphoton spectrometer (Olympus, Japan).The Bruker EMXnano was used for EPR testing of the molecule.Two-photon laser (Olympus, Japan) was used for the cell phototoxicity and the effect of photodynamic therapy on living tumours.The independent ventilated cage box system (IVC, Xinhua Medical Device Co., LTD.) was used for mouse feeding.

Spectrometric determination in vitro
An ultraviolet spectrophotometer (GBC Scientific Equipment Pty LTD, Australia) and fluorescence spectrophotometer FluoroMax-4 (HORIBA, Japan) were used to measure the absorption spectra and fluorescence spectra, respectively.In all spectral experiments, the final solutions contained < 5 ‰ DMSO.Each experiment was carried out in five replicates (n = 5).Synthesis of intermediate ZZ. 4-bromo-1, 8-naphthalene anhydride (0.5 g, 1.0 eq.) was added to a double-mouth round-bottom flask with 20 mL anhydrous ethanol, then ethylenediamine (145 μL, 1.2 eq.) was added.The reaction mixture was stirred at 80 °C for 1.0 h until 4-bromo-1, 8-naphthalene anhydride was completely reacted (monitoring via TLC).After that, it was poured into 20 mL of ice water and the resulting suspension filtered, dried to obtain the crude product ZZ.Then the crude products were purified by column chromatography using dichloromethane / methanol (100:0 to 100:8, v/v) to obtain ZZ as white solid.

Synthesis of ZZ, ZZ-Br
Yield 65% (0.37 g).Synthesis of photosensitive dye ZZ-EDA.ZZ-Br (0.30 g, 1.0 eq.) and anhydrous potassium carbonate (0.44 g, 5.0 eq.) were added to a double-mouth round-bottom flask with 25 mL of anhydrous ethanol then stirred and heated to 80 °C for 1.0 h.Then ethylenediamine (0.38 g, 10.0 eq.) was added into the reaction system which was then continued to be stirred at 80 °C for 6.0 h until ZZ-Br was completely reacted (monitoring via TLC).Then the solvent of the reaction mixture is evaporated to obtain the crude product.Then the crude product was purified by column chromatography using dichloromethane / methanol (100:0 to Synthesis of photosensitive dye ZZ-NN.ZZ-Br (0.30 g, 1.0 eq.) was added to a round-bottom flask with 10 mL of anhydrous pyridine.Subsequently, 200 μL triethylamine was added into the reaction mixture.After stirred and heated to 120 °C for 1.0 h, then ethylenediamine (0.84 g, 15.0 eq.) was added into the reaction system which was then continued to be stirred at 120 °C for 2.0 h until ZZ-Br was completely reacted (monitoring via TLC).Then the solvent of the reaction mixture is evaporated with cyclohexane to obtain the crude product.Then the crude product was purified by column chromatography using dichloromethane/methanol (100:0 to 100:10, v/v) to obtain ZZ-NN as a yellow solid, Yield 63 % (0.19 g).Synthesis of photosensitive dye ZZ-NH 2 .4-bromo-1, 8-naphthalene anhydride (0.5 g, 1.0 eq.) was added to a double-mouth round-bottom flask with 15 mL butyl oxitol, then hydrazine hydrate (880 μL, 10 eq.) was added.The reaction mixture was stirred at 120 °C for 4.0 h until 4-bromo-1, 8-naphthalene anhydride was completely reacted under the protection of nitrogen.
After that, it was cooled to room temperature, yellow precipitate was precipitated, filtered, and washed with water 3 times to obtain the crude product.Then the crude product was recrystallized with DMF/acetonitrile (1:1, v/v) to obtain the product ZZ-NH 2 .Yield 92% (0.37 g).

Measurement of relative fluorescence quantum yield
The photosensitive dyes ZZ-EDA and ZZ-NN were diluted in different pH buffers (pH = 5.0 or pH = 7.2) with a final concentration of 20 μM, respectively.The different pH buffer solution is a mixture of 2.0 mL phosphoric acid buffer at pH = 5.0 or pH = 7.2 and 1.0 mL methanol.Then the absorption spectra and fluorescence spectra were measured respectively, and the slit settings of fluorescence spectra were consistent.The obtained data were calculated using the following formula to obtain the fluorescence quantum yields of ZZ-EDA and ZZ-NN.
Note: Φ represents fluorescence quantum yield; F represents the integral area of the fluorescence emission spectrum minus the background fluorescence; A represents the absorbance value at the maximum absorption wavelength; λ ex represents the maximum excitation wavelength; n is the refractive index of different solutions; x and s represent unknown and standard samples, respectively.Fluorescein was chosen as the standard reference.All the experimental results obtained were from five parallel experiments.

Photostability test
Prepare ZZ-EDA and ZZ-NN with a final concentration of 3.0 μM using phosphate buffer solution (pH = 7.4) as the solvent.
Place them at a distance of 30 cm from a 500 W tungsten iodide lamp to irradiate it continuously for 5.0 h.The saturated NaNO 2 aqueous solution acts as a light filter and a thermal filter is placed between the sample and the light source to filter light less than 400 nm and most of the heat.The change of fluorescence intensity under different illumination times was measured.
The setting of fluorescence spectrum parameters was consistent during the measurements.

Detection of 1 O 2 production in buffer solutions of different pH
Compound 9,10-anthracenediyl-bis(methylene)-dimalonic acid (ABDA) was used as indicator for detection of 1 O 2 in buffer solutions (pH = 7.2, PBS buffer).When 1 O 2 is generated in the system, the ABDA will be oxidized and the absorption at 400 nm decrease.ZZ-NN (60 µmol) and ABDA (120 µmol) are dissolved in 3.0 mL buffer solution.The mixture was then placed in a cuvette and irradiated.The wavelength of irradiation light is 800 nm with 9.15 mW cm -2 .The absorption change of sample at 400 nm was recorded by the UV-Vis absorption spectrophotometer.The production of 1 O 2 of ZZ-Br, ZZ-NH 2 and ZZ-EDA in different pH solution was detected using the same method.

TCNQ quenching experiment
The compound tetracyanoquinodimethane (TCNQ) was used as the quencher (electron acceptor) in buffer solutions of different pH (pH = 5.0 and pH = 7.2, PBS buffer).ZZ-NN (60 μmol) was dissolved in 3.0 mL buffer solution with a pH of 5.0 or 7.2.
Then TCNQ (6.0 mM) was added in equal amounts (20 μL) in turn and the fluorescence intensity at 533 nm was monitored.
The TCNQ experiment of ZZ-EDA was carried out in the same way.

Cell culture
HepG2 cell lines and 4T1 cell lines were obtained from the Chinese Academy of Medical Sciences.The medium for the cells were made up of phenol red-free Dulbecco's Modified Eagle's Medium (DMEM, WelGene), and 10% fetal bovine serum (FBS; Gibco) and 1% penicillin/streptomycin.The two kinds of cell lines were all grown in a CO 2 incubator at 37 °C.One day before imaging, the cells mentioned above were seeded into confocal dishes with well glass bottom (MatTek, 1# glass, 0.13-0.16mm).
They were incubated at 37 °C in 5.0 wt %/vol CO 2 for 24 h.Then, the cells were incubated with the photosensitive dyes (ZZ-EDA and ZZ-NN) at a certain concentration, respectively.

ROS production in living cells
HepG2 cells were seeded in confocal dishes and incubated for 24 h.Specifically, the HepG2 cells were incubated with 5.0 μM photosensitive dyes (ZZ-EDA, ZZ-NN) at 37 °C for 1.0 h and then washed with PBS buffer three times.The culture medium was then replaced with DMEM containing 6 μM DCFH-DA and incubated for 30 min.After that, the DMEM was removed and the remaining DCFH-DA was washed three times with PBS buffer.The petri dishes were irradiated (800 nm, 9.15 mW cm −2 ) for 3.0 min, and the fluorescence signal of DCFH-DA in cells was captured by Olympus FV1200 laser confocal microscope with a 60× objective lens.

O 2 •-production in living cells
HepG2 cells were seeded in confocal dishes and incubated for 24 h.Specifically, the HepG2 cells were incubated with 5.0 μM photosensitive dyes (ZZ-EDA, ZZ-NN) at 37 °C for 1h and then washed with PBS buffer three times.And the culture medium was replaced with DMEM containing 10 μM DHE and incubated for 30 min.After that, the DMEM was removed and the remaining DHE was washed three times with PBS buffer.The petri dishes were irradiated (800 nm, 9.15 mW cm −2 ) for 3 min, and the red fluorescence signal formed when oxidized DHE binds to DNA in cells was captured by Olympus FV1200 laser confocal microscope with a 40× objective lens.

HepG2 mitochondrial membrane potential in PDT experiment
The mitochondrial membrane potential assessment experiment in HepG2 cells was conducted using by MitoProbe DiIC1(5) probe.HepG2 cells cultured in glass-bottom confocal dishes overnight were incubated with photosensitive dyes (ZZ-EDA, ZZ-NN, 5.0 μM) in DMEM medium for 1 h.Then cells were washed with PBS three times and were irradiated (800 nm, 9.15 mW cm -2 ) for 3.0 min or 6.0 min, respectively.After washing with PBS, cells were stained with MitoProbe DiIC1(5) (5.0 μM) at 37 °C for 30 min.Then cells were washed with PBS and replaced with fresh DMEM.Finally, the fluorescence signal of MitoProbe DiIC1(5) in cells were taken by Olympus FV1200 laser confocal microscope with a 60×objective lens.The mean fluorescence density of the red fluorescence channel (648-688 nm) was calculated by FV10-ASW 4.2 Viewer.

Calcein AM/PI staining of HepG2 cells in PDT experiments
The HepG2 cells were seeded on confocal dishes and incubated for 24 h.The medium was then replaced with fresh culture medium containing different photosensitive dyes (ZZ-EDA, ZZ-NN, 5.0 μM) and incubated for 1 h.After that, the cells were irradiated (800 nm, 9.15 mW cm −2 ) for 6.0 min and were incubated for 2.0 h.Then, the culture medium was replaced with DMEM containing 4.5 μM propidium iodide (PI).After further incubation for 30 min, 2.0 μM of Calcein AM was added for 5.0 min incubation.Then the calcein AM and PI solution was removed and washed with PBS buffer three times.Fluorescent images of Calcein AM and PI, staining on the cells, were promptly captured by Olympus FV1200 laser confocal microscope with a 40× objective lens.

In vivo PDT experiment
In this work, all animal experiments involved have been approved by the local research ethics review board of the Animal Ethics Committee of the Xinxiang Medical University (Henan, China, ethics statement Reference No. 2015016).And all the mice were used in accordance with institutional ethics committee regulations and guidelines on animal welfare.
Phototoxicity assay was performed by using 4T1 tumour bearing mice (BALB/c mice for the PDT experiment).The mice were divided into four different groups for treatment: Group 1: blank group (without any treatment); Group 2: irradiated group (light irradiation only); Group 3: ZZ-NN group (ZZ-NN injection without irradiation); Group 4: ZZ-NN+Light group (ZZ-NN injection with irradiation).Each group contained five mice.The injection method of photosensitive dye is intratumoural injection.30 min after injection of ZZ-NN, the tumour region of groups 2 and 4 were irradiated (800 nm laser, 20.10 mW cm -2 ) for 30 min.The tumours were treated with injection and light at days 1, 3 and 5.The effect of the different treatment groups was monitored by measuring tumour size (tumour size = width × width × length / 2.) and mice body weight for 14 days after PDT treatment.After 14 days, the tumours were dissected and photographed.Tumour tissues of the treated group and untreated group were harvested for histological study by hematoxylin-eosin (H&E) staining.

Supplemental figures
Scheme S1.The synthetic route of ZZ-EDA and ZZ-NN.
Dihydroethidium (DHE) was used as an indication for detection of O 2 •-in PBS (pH = 7.2).The oxidized product of DHE by O 2 •-could intercalate into DNA to emit red fluorescence.ZZ-NN (60 μmol), DHE (60 μmol) and DNA were dissolved in 3.0 mL buffer solution.The mixture was placed in a quartz cuvette and illuminated.The wavelength of irradiation light is 800 nm with 9.15 mW cm -2 .The change of fluorescence intensity at 610 nm was recorded by fluorescence spectrophotometer.The production of O 2 •-of ZZ-Br, ZZ-NH 2 and ZZ-EDA in different pH solution was detected using the same method.The compound dihydrorhodamine 123 (DHR 123) was also used as an indicator for detection of O 2 •-in buffer solutions of different pH (pH = 5.0 and pH = 7.2, PBS buffer).Non-fluorescent substance DHR 123 can be oxidized by O 2 •-and emits fluoresce at 525 nm.ZZ-NN (60 μmol) and DHR 123 (60 μmol) were dissolved in 3.0 mL buffer solution with a pH of 5.0 or 7.2.The mixture was placed in a quartz cuvette and illuminated.The wavelength of irradiation light is 800 nm with 9.15 mW cm -2 .The change of fluorescence intensity at 525 nm was recorded by fluorescence spectrophotometer.The production of O 2 •- of ZZ-Br, ZZ-NH 2 and ZZ-EDA in different pH solution was detected using the same method.

Figure
Figure S5. 1 H NMR spectrum of ZZ-Br.

Figure
Figure S7. 1 H NMR spectrum of ZZ-NN.

Figure S17 .Figure S18 .
Figure S16.pH interference and photo stability test of ZZ-EDA and ZZ-NN.The change of fluorescence intensity at the maximum emission (530 nm) of (a) ZZ-Br, (b) ZZ-NH 2 , (c) ZZ-EDA (3.0 μM) in different pH solutions (a mixed solution of PBS at different pH and methanol, V:V = 2:1).The change of fluorescence intensity at the

Figure
Figure S22.EPR spectra of ZZ-Br, ZZ-NH 2 , ZZ-EDA with or without light irradiation (TEMP was used as a trapping agent for 1 O 2 ; DMPO was used as a trapping agent for O 2 •− ).

FigureFigure S24 .Figure S25 .Figure S26 .
Figure S23.(a) Electron-hole distributions of the S 1 states of protonated ZZ-NN, and ZZ-EDA.The green regions represent electrons and the blue regions represent holes.(b) The difference between singlet state (S 1 ) and triple state (T 1 ) energy levels of protonated ZZ-NN, and ZZ-EDA, respectively.(c) The protonation process of ZZ-NN and ZZ-EDA was monitored by TLC, respectively.

Figure S29 .Figure S30 .
Figure S29.Diffusion experiment of ZZ-NN in vivo.Fluorescence imaging of 4T1-tumour-bearing mice at wavelength range of 500-560 nm by ZZ-NN at different time.The histogram shows tumour fluorescence intensity extraction at different time.